CN107438668A - Utilize the pluripotent cell apparatus for deivation and method of energy - Google Patents
Utilize the pluripotent cell apparatus for deivation and method of energy Download PDFInfo
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Abstract
The pluripotent cell apparatus for deivation and method of energy are the present invention relates to the use of, in more detail, applies the energy of ultrasonic wave, laser or heat treatment etc., Novel multifunctional cell of the induction with versatility to differentiated cell.
Description
Technical field
The pluripotent cell apparatus for deivation and method of energy are the present invention relates to the use of, by providing at ultrasonic wave, laser or heat
The energy such as reason can induce the pluripotent cell with versatility.
Background technology
Versatility (pluripotency) is to be divided into 3 germinal layer systems, i.e. epiblast, mesoderm and the ability of endoderm.
Multipotential stem cell is clinically important in disease model and transplanting, because it forms arbitrary cell or tissue in vivo
Type.Therefore, at present embryonic stem cell, inductive pluripotent stem cells (induced pluripotent stem cell,
IPSC), body cell and demand main in the reprogramming or differentiation of patient's derived cell be, for clinical practice, it is not necessary to draw
Enter external inhereditary material or chemical substance or small molecule, it is necessary to simple, quick, effective and safe.In nearest research,
Demonstrate interaction between environment and genotype and the gene expression of Living Organism and phenotypic variation is closely related.Pass through tune
The environmental stimuluses such as nodule structure, machinery, magnetic, ultrasonic signal can adjust cell death, propagation and cell absorption efficiency.With this
Correctly molecular mechanism is also uncertain similar in a little methods, therefore these methods can only be used as and need not introduce inhereditary material, change
Learning material and small molecule can realize that the scheme of functioning in an acting capacity of of security receives.
At this point, the present inventor is under conditions of no gene and chemical substance, by using cellular environment
The energy of signal, which provides, makes non-differentiation marker thing and 3 germinal layer marker gene tables being made up of epiblast, mesoderm and endoderm
Reaching, exploitation is induced to differentiate into the Novel multifunctional cells with versatility of 3 germinal layers, i.e. Physics (pluripotent
sphereyielded by ultrasonic sTimulus) the method for cell.
The content of the invention
Technical scheme
It is an object of the invention to provide a kind of pluripotent cell abductive approach and apparatus for deivation, it is not necessary to differentiated cell
Inverse differentiation induction factor and chemical substance are introduced, induces differentiated Hemapoiesis that there are the new more of versatility by providing energy
Can cell.
Technological means
To achieve these goals, the present invention is provided by the inverse method for being divided into pluripotent cell of differentiated cell, including is mixed
Culture medium and differentiated cell are closed, provides energy to the mixture, and orbicule is formed by the culture of a period of time
(spheroid), the orbicule has versatility.
The present invention also provides pluripotent cell apparatus for deivation, including can store the culturing room of cell and culture medium；And setting
, can be to the cell and the power supply device of culture medium offer energy in the side of the culturing room；Mix differentiated cell and
Culture medium, energy is provided to the mixture, and orbicule is formed by the culture of a period of time, the orbicule has multipotency
Property.
Technique effect
The present invention need not introduce inverse differentiation induction factor and chemical substance to differentiated cell, pass through suitable ultrasound
The energy of the offers such as ripple, laser or heat treatment induce differentiated Hemapoiesis have versatility and with the more work(of known inductivity
Can the stem cell Novel multifunctional cell stronger compared to differentiation characteristic.
Brief description of the drawings
Fig. 1 is the simulation drawing of mankind's Physics cells with versatility of the present invention；
Fig. 2 a show effect of the ultrasonic intensity to human dermal fibroblasts, a) under more different ultrasonic intensities
HDF metamorphosis, b) number of many cells orbicule that generates under different ultrasonic intensities.
Fig. 2 b show effect of the ultrasonic intensity to human dermal fibroblasts, c) HDF of ultrasonication work/
Dead cell analysis result, d) it is above-mentioned c) in existence and damaging cells ratio.
Fig. 3 a are shown in 1W/cm2Fixing intensity under ultrasonic exposure time effect, a) more different ultrasonic waves are sudden and violent
Reveal the HDF metamorphosis under the time, b) number of many cells orbicule that generates under different ultrasonic exposure times.
Fig. 3 b are shown in 1W/cm2Fixing intensity under ultrasonic exposure time effect, c) ultrasonication HDF
Live/dead cell analysis result, d) it is above-mentioned c) in existence and damaging cells ratio.
Fig. 4 shows effect of the ultrasonic intensity to mankind's ESC culture mediums, and the figure of upper end is compared in ultrasonication
The HDF of the ultrasonication grown in culture medium metamorphosis, the figure of lower end show generated in above-mentioned culture medium it is more
The number of spheroids.
Fig. 5 is shown in 5W/cm2Fixing intensity under ultrasonic exposure time to the effects of mankind's ESC culture mediums, a) compare
Compared with the HDF of the ultrasonication grown in the culture medium of ultrasonication metamorphosis, b) in the more of above-mentioned a) middle generation
The number of spheroids.
Fig. 6 is shown for generating the ultrasonication condition of many cells orbicule and the effect of condition of culture, a) is suspended
Culture, b) monolayer cultivation.
Fig. 7 a show that many cells of different ultrasonic stimulation (UC, UM, the UCUM) generations under condition of suspension culture are spherical
The Size Distribution of body, a) overall dimensions be distributed, b) 50-100 μ m in size orbicule distribution.
Fig. 7 b show that many cells of different ultrasonic stimulation (UC, UM, the UCUM) generations under condition of suspension culture are spherical
The Size Distribution of body, c) 100-200 μm, d)>The orbicule distribution of 200 μ m in size.
Fig. 8 a show that many cells of different ultrasonic stimulation (UC, UM, the UCUM) generations under monolayer culture conditions are spherical
The Size Distribution of body, a) overall dimensions be distributed, b) 50-100 μ m in size orbicule distribution.
Fig. 8 b show that many cells of different ultrasonic stimulation (UC, UM, the UCUM) generations under monolayer culture conditions are spherical
The Size Distribution of body, c) 100-200 μm, d)>The orbicule distribution of 200 μ m in size.
Fig. 9 a show the pluripotency marker's thing for comparing the mankind's Physics cells under culture and monolayer culture conditions that suspend
The RT-PCR analysis results of gene, OCT3/4 (a) and SOX2 (b) expression.
Fig. 9 b show the pluripotency marker's thing for comparing the mankind's Physics cells under culture and monolayer culture conditions that suspend
The RT-PCR analysis results of gene, NANOG (c) and TDGF1 (d) expression.
The laser scanning co-focusing microscope image of OCT3/4 expressions when Figure 10 a are analysis suspension cultures.
Figure 10 b are the laser scanning co-focusing microscope images of OCT3/4 expressions when analyzing monolayer cultivation.
Figure 11 show culture 6 days during pluripotency marker's thing gene expression RT-PCR analysis results.
Figure 12 shows RT-PCR analysis results, determines under different incubation times in the HDF orbicules of ultrasonication
OCT3/4 expression opportunity.
Figure 13, which is shown, determines representational non-differentiation marker thing expression in ES cells, HDF and mankind's Physics cells
As a result.
Figure 14 shows the alkaline phosphatase staining result of the pluripotent state for characterizing many cells orbicule.
Figure 15 shows the immunocytochemistry result of pluripotency marker's thing expression.
Figure 16 a show the facs analysis result of mankind ES (H9) cell surface marker thing.
Figure 16 b show the facs analysis result of mankind's HDF cell surface marker things.
Figure 16 c show the facs analysis result of mankind's Physics cell surface marker things.
Figure 17 is shown when co-incubation raises (Feeder) cell and Physics cells, is analyzed using RT-PCR more
The gene expression (a) of energy property label and the result using immunocytochemical method analysing protein expression (b).
Figure 18 shows sodium hydrogensulfite sequencing result, shows the methylation state of COT3/4 and NANOG promoters.
Figure 19 shows the multiplication capacity the result of mankind's Physics cells, a) shows proliferation marker Ki67's
Expression, b) show the result that increased cell is dyed using H33342 and PI, c) show orbicule under different incubation times
Size.
Figure 20 shows the experimental result of the self-regeneration ability of checking mankind's Physics cells.
Figure 21 a show that mankind ES (H9) cell expresses the immunocytochemical assay result of 3 germinal layer labels.
Figure 21 b show that mankind Physics cells express the immunocytochemical assay result of 3 germinal layer labels.
Figure 22 showed culture mankind Physics cells during 15 days, the expression mould about OCT3/4 and 3 germinal layer labels
The immunocytochemical assay result of formula.
Figure 23 shows the SEM image of mankind's Physics cells under the conditions of different ultrasonic stimulations.
Figure 24 is shown carry out under the conditions of the different ultrasonic stimulations after ultrasonication and cultivate 2 hours after, utilize
The live/dead cell analysis result of mankind's Physics cells shown in fluoroscopic image.
Figure 25 is shown to form many cells orbicule after, the live/dead cell analysis result of mankind's Physics cells.
Figure 26 shows intracellular Ca caused by ultrasonic stimulation2+The change of concentration.
Figure 27 is shown because of the H of ultrasonic stimulation generation2O2Analysis result.
Figure 28 be in order in analysis chart 27 because ultrasonication generation intracellular H2O2, with the people of CM-H2DCFDA dyeing
The fluoroscopic image of class Physics cells.
Figure 29 shows the analysis result of intracellular ATP releases caused by ultrasonic stimulation.
Figure 30 shows the expression pattern of P2X and P2Y acceptors in mankind's Physics cells.
Figure 31 is the Laser Scanning Confocal Microscope figure that the higher penetrating power of mankind's Physics cells is determined using quantum dot 705
Picture.
Figure 32 shows the RT- for expressing pluripotency marker's thing gene by allochthon in mankind's Physics cell culture mediums
PCR analysis results.
Figure 33 is immunocytochemistry result, shows that the allochthon OCT4 of mankind Physics cells release transmits to HDF
Process, yellow arrows represent periphery HDF in OCT3/4 expression.
Figure 34 shows the Tri-labeling method of 3 germinal layer marker representations of mankind's Physics cells of vitro differentiation induction
Learn result.
Figure 35 shows the RT-PCR of the nervous system gene expression of the vitro differentiation for determining mankind's Physics cells
Analysis result.
Figure 36 shows the RT-PCR of the cardiac system gene expression of the vitro differentiation for determining mankind's Physics cells
Analysis result.
Figure 37 shows the immune of the nervous system marker representation of the vitro differentiation for determining mankind's Physics cells
Cytochemistry result.
Figure 38 shows the immune of the cardiac system marker representation of the vitro differentiation for determining mankind's Physics cells
Cytochemistry result.
Figure 39 is the immunocytochemistry image of actinine.
Figure 40 shows the karyotyping result of mankind's Physics cells of the present invention.
Figure 41 shows that the histogenic immunity fluorescence for determining mankind's Physics cells differentiation in vivo in mouse testis contaminates
Color result.
Figure 42 shows glimmering for the histogenic immunity that determines mankind Physics cells internal muscle differentiation in mouse thigh
Light coloration result.
Figure 43 shows the effect of cell culture medium, a) utilizes the result of ES culture mediums and HDF culture mediums, b) cultivated in ES
Mankind's Physics cells are induced to determine the result of ES labels in base and HDF culture mediums.
Figure 44 shows the result that mankind's Physics cells are induced in other cell lines, a) shows cellular morphology
Change, b and c are to induce mankind's Physics cells to determine b) ES labels and c) 3 germinal layer label in other cell lines respectively
Result.
Figure 45 is shown by the result of patient skin cell induction mankind's Physics cells, a) shows cellular morphology
Change, b) induction mankind's Physics cells determine the result of ES labels and 3 germinal layer labels in other cell lines.
Figure 46 show using other energy sources carry out heat treatment induction mankind's Physics cells after determine ES labels and
The result of 3 germinal layer labels.
Figure 47, which shows to use a laser as after other energy sources induce mankind's Physics cells, determines ES labels and 3
The result of germinal layer label.
Figure 48 shows the process of the inducing mouse Physics cells in MEC.
After Figure 49 shows MEC (OG2MEF cells) the progress ultrasonication to conversion, it is determined that
The change of cellular morphology and Oct4 expression under different incubation times, A, which is shown, determines GFP in mouse Physics orbicules
The result of expression, B are that the image synthesized after multiple images of wide scope is shot using tile scan (Tile scan).
Figure 50 show by ultrasonic waveform into orbicule GFP expression rates chart and using flow cytometry analysis
GFP expression rates in the total cells of ultrasonication.
Figure 51 shows the cell surface non-differentiation marker thing of the mouse Physics cells using flow cytometry analysis
(SSEA1) result of expression.
Figure 52 shows the RT-PCR analysis results of pluripotency marker's thing gene of mouse Physics cells.
Figure 53 shows the versatility protein label for the mouse Physics cells analyzed by immunocytochemical method
Result.
Figure 54 shows the alkaline phosphatase staining result of the pluripotent state for characterizing mouse Physics cells.
Figure 55 shows the RT-PCR analysis results that 3 germinal layer labels are expressed about mouse Physics cells.
Figure 56 shows the immunocytochemical method analysis result that 3 germinal layer labels are expressed about mouse Physics cells.
Figure 57 shows the karyotyping result of mouse Physics cells.
Embodiment
Hereafter the present invention is specifically described.
The present invention relates to by the inverse method for being divided into pluripotent cell of differentiated cell, including mixed culture medium and differentiated thin
Born of the same parents, energy is provided to the mixture, and orbicule (spheroid), the orbicule tool are formed by the culture of a period of time
There is versatility.
The present invention need not introduce the inverse induction material such as inverse differentiation induction factor, chemical substance to differentiated cell,
By provide suitable energy induce differentiated Hemapoiesis have versatility and with known inductivity versatile stem cell
The Novel multifunctional cell stronger compared to differentiation characteristic.
Above-mentioned pluripotent cell under external environment condition can induction well, with progenitor cells (progenitor cell)
Similar, differentiation characteristic is more stronger than stem cell, therefore has difference with existing inductive pluripotent stem cells.That is, when with induction
, it is necessary to be undergone the standard of atomization to a certain degree when the property similar embryonic stem cell of multipotential stem cell is as cellular therapeutic agent
In the standby stage, consider that the dangerous key element of cancer may be changed into, and viral vector used in the inverse differentiation induction factor of introducing can draw
Rise safety issue, and the present invention pluripotent cell, it is not necessary to introduce in addition for genetic mutation inverse differentiation induction factor or
The inverse induction material such as chemical substance can induce, it is not necessary to other kinds of cell co-culture, therefore not have cell
Pollution problem (with other mixing with cells), in testing in vivo, does not form the teratoma similar with cancer cell, therefore do not have cancer
The problem of generation, it can be ensured that security.In other words, it is that Induction Process is simple and uses the advantages of pluripotent cell of the invention
When it is few, can significantly shorten from processing autogenous cell to transplanting time.
The present invention specifically provides energy to both culture medium and differentiated cell, therefore can be given birth to outstanding yield
Glomeration body.
Above-mentioned energy can be any one in ultrasonic wave, laser or heat treatment.
The present invention pluripotent cell the characteristics of be can stably express OCT3/4, SOX2, NANOG, c-MYC, KLF4,
Any one undifferentiated mark in TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4 or AFP
Note thing or 3 germinal layer marker genes being made up of mesoderm and endoderm.
Pluripotent cell can be generated based on described above need not being introduced to differentiated cell against differentiation induction factor
Viewpoint, the present inventor consider its relation between excretion body.That is, ultrasonic wave, laser or heat treatment can pass through
Energy inducing temperature rises, induced activity oxygen (ROS, reactive oxygen species) produces, and induction is given birth to by ultrasonic wave
Into the vibration of microbubble, induced fluid flowing, i.e. induction generates microstrip along cell membrane, so as in cell membrane
It is upper to produce small damage, the formation in hole is induced, so as to increase the absorption to exterior materials, this can be intracellular by analyzing
Ca2+Change in concentration and determination H2O2Whether generate line justification.That is, intracellular Ca is analyzed2+Change in concentration, there is damage in cell membrane
Or film mobility increase when reaction be intracellular (cytosol) Ca2+The moment increase of concentration, so that it is determined that cell membrane flow
Property increase.According to one embodiment of present invention, after ultrasonication, Ca2+Concentration quickly increases at once, then gradually subtracts
It is small, the level of the control group by being decreased to not carry out ultrasonication, it may be determined that carried out after inducing cell membrane damage extensive
It is multiple.In addition, determine intracellular H2O2The experiment whether generated and similar pattern, after initial stage ultrasonication, H2O2Generation
Amount increases immediately, is then gradually decrease to control group level, is recovered after may thereby determine that inducing cell membrane damage.Separately
Outside, by the use of ATP as to various kinds of cell stress reaction signal, analyze the ATP concentration in pluripotent cell after ultrasonication
Result, compared with undressed control group, with higher horizontal release ATP.Meanwhile promote ion caused by ATP discharges
The expression of type P2X acceptors and metabolic P2Y acceptors is also more active than in control group in pluripotent cell.
On the other hand, it is known that hereditary information material (DNA, mRNA, microRNA, protein) is included inside excretion body, because
The excretion body that cell membrane damage is discharged to cell membrane exterior is again introduced into other cells on periphery, can be passed by this process
Pass hereditary information material existing for inside excretion body.Therefore, induce or even promote thin using being stimulated caused by ultrasonication
Intracellular low expression or the expression for maintaining the non-differentiation marker thing for expressing the state being suppressed, while damage is produced on cell membrane
Wound, excretion body existing for the cell interior for the non-differentiation marker thing for making to be induced or even promoted comprising above-mentioned expression are discharged to outer
Portion, so as to peritropous cell transmission, because the cell on periphery is also the state that cell membrane fractions sustain damage, so speculating cell
Membrane fluidity increase, the efficiency that excretion body enters cell interior are higher than normal condition, it is believed that existing for inside this excretion body
The undifferentiated relevant genetic information that above-mentioned expression is induced or even promoted is passed so as to generate pluripotent cell.The one of the present invention
In individual specific embodiment, culture medium is reclaimed in pluripotent cell Induction Process, extracts the excretion body in culture medium, confirms excretion body
Inside whether there is the result of the non-differentiation marker thing related to pluripotent cell, find and non-differentiation marker thing known to determining
Expression is high, so as to support the above-mentioned hypothesis of inventor.Find simultaneously under above-mentioned ultrasonic wave, laser or heat treatment,
Caryogram is also normal, is not made a variation.
Above-mentioned hypothesis makes it possible to induce the discharge of excretion body to generate pluripotent cell by cell membrane damage.
In this manual, term " pluripotent cell " refers to, by energy, strictly, pass through ultrasonic wave, laser or heat
Pluripotent cell is obtained after processing.In this manual, versatility is to instigate the non-differentiation marker thing expressed in embryonic stem cell
The state of stable expression.Meanwhile and instigate 3 germinal layer marker representations of the endoderms of three species, epiblast and mesoderm
State.Above-mentioned pluripotent cell can with " Physics (pluripotent sphere yielded by ultrasonic sTimulus) cell " or " Physics orbicules " are mixed.Hereinafter with reference to Fig. 1 to the present invention by the inverse differentiation of differentiated cell
It is described in detail for the method for pluripotent cell.
First, mixed cell culture base and differentiated cell, energy is provided to said mixture.
Before energy is provided to the mixture of above-mentioned differentiated cell, first energy, Ke Yiti are provided to cell culture medium
The high inverse efficiency for being divided into pluripotent cell.
Above-mentioned energy can be any one in ultrasonic wave, laser or heat treatment.
The ultrasonication carried out to above-mentioned culture medium can be that output intensity is 1W/cm2To 20W/cm2Ultrasonic wave at
Reason 1 minute to 20 minutes, specifically, output intensity 2W/cm2To 10W/cm2Ultrasonication 5 minutes to 15 minutes, more
Body, output intensity 3W/cm2To 7W/cm2Ultrasonication 7 minutes to 13 minutes.
The laser treatment carried out to culture medium can be that the pulsed laser beam of 300nm to 900nm wave bands irradiates 1 second to 20
Second, specifically, the pulsed laser beam of above-mentioned wave band irradiates 3 seconds to 10 seconds, more specifically, the pulsed laser beam of above-mentioned wave band
Irradiation 4 seconds to 6 seconds.For example, above-mentioned wave band can use 400nm, 808nm, 880nm wavelength.
The heat treatment carried out to culture medium can be heat-treated 5 minutes to 20 points under 40 DEG C to 50 DEG C of temperature conditionss
Clock.
Above-mentioned culture medium can use embryonic stem cell medium, stem cell induction culture medium etc..
Above-mentioned differentiated cell can use the fibroblast of mammal source；Including cervical cancer cell (HeLa
Cell the cancer cell including), or organ inner tissue cell including pulmonary epithelial cells (L132cell) etc..
When providing energy to differentiated cell, it is preferably exposed under some strength, if departing from this scope, cells survival
Rate may be reduced.
Therefore, the ultrasonication carried out to the mixture of culture medium and differentiated cell can be that output intensity is
0.5W/cm2To 3W/cm2Processing 1 second to 5 seconds, specifically, output intensity 0.7W/cm2To 2W/cm2Processing 1 second to 5 seconds, more
Specifically, output intensity 0.8W/cm2To 1.5W/cm2Processing 1 second to 5 seconds.
The laser treatment carried out to the mixture of culture medium and differentiated cell can be arteries and veins of the 300nm to 900nm wave bands
Laser beam is rushed to irradiate 1 second to 20 seconds, specifically, the pulsed laser beam of above-mentioned wave band irradiates 3 seconds to 10 seconds, more specifically,
The pulsed laser beam of above-mentioned wave band irradiates 4 seconds to 6 seconds.For example, above-mentioned wave band can use 400nm, 808nm, 880nm
Wavelength.
The heat treatment carried out to the mixture of culture medium and differentiated cell can be under 40 to 50 DEG C of temperature conditionss
After exposure 1 minute to 10 minutes, exposure 5 seconds to 10 seconds under 0 DEG C to 4 DEG C of temperature conditionss.
Afterwards, the culture of certain time is carried out to the mixture for providing energy, forms the orbicule with versatility.
The culture carried out to the mixture for providing energy can be by culture (suspended culture) or the individual layer of suspending
The mode of (monolayer culture) is cultivated, it is 3 to allow the time that the orbicule of the stable expression of non-differentiation marker thing is formed
It was to 10 days.Simply, because whether the different versatility orbicules of the species of training method, cell or culture medium form also difference,
So those skilled in the art can suitably adjust above-mentioned incubation time according to actual conditions.
According to the specific embodiment of the present invention, compared with monolayer cultivation, the orbicule formation efficiency for the culture that suspends is more
It is high.Also, compared with monolayer cultivation, the orbicule number and size for the culture that suspends are bigger, there is certain Size Distribution.
According to the specific embodiment of the present invention, enter in the human dermal fibroblasts to ultrasonic wave or laser treatment
During row suspension culture, since the about the 3rd day, it was observed that the expression increase of non-differentiation marker thing or stabilisation, inverse from this point on
Differentiation starts.In addition, when the human dermal fibroblasts to heat treatment suspend culture, since the about the 8th day, observation
Expression increase or stabilisation to non-differentiation marker thing, from this point on inverse differentiation start.
By non-differentiation marker thing, for example, OCT3/4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-
The expression of 60 grades, it may be determined that orbicule has versatility.The determination of non-differentiation marker thing can be thin by RT-PCR or immune
Born of the same parents' chemical method is analyzed, and this is had no particular limits.
In addition, the pluripotent cell of the present invention makes 3 germinal layer labels, i.e. epiblast (PAX6, Nestin), mesoderm
(Brachyury, SMA), endoderm (GATA4, AFP) label are expressed at high levels.
In addition, the pluripotent cell of the present invention expresses proliferation marker albumen Ki-67, there is multiplication capacity.
In addition, when the pluripotent cell of above-mentioned inverse differentiation is with vegetative cell co-incubation, the propagation increase of pluripotent cell can be with
It is determined that after being cultivated in induction culture medium, epiblast/endoderm/mesoderm and nerve cell/cardiac muscle cell are divided into.
The present invention also provides pluripotent cell apparatus for deivation, including can store the culturing room of cell and culture medium；And setting
In the side of the culturing room, the power supply device of energy can be provided to the cell and culture medium, mix differentiated cell and
Culture medium, energy is provided to the mixture, and orbicule is formed by the culture of a period of time, the orbicule has multipotency
Property.
Above-mentioned culturing room refers to the incubator used in conventional cell culture.For example, culturing room has temperature control
Portion and carbon dioxide control unit, those skilled in the art can be suitably adjusted in culturing room according to purpose and the species of cell
Cell culture condition.
In addition, because differentiated cell can be made thin against multipotency is divided into by way of suspend culture or monolayer cultivation
Born of the same parents, so, this culture is turned into possible structure it is preferred that above-mentioned culturing room has.For example, it may be for the culture that suspends
The culturing room with agitator.
The above-mentioned power supply device that can provide energy can include the ultrasonic generator of transmitting ultrasonic wave, launch laser
Generating device of laser, or temperature control equipment.
Above-mentioned ultrasonic generator can produce the existing ultrasonic wave that frequency is 10kHz to 100MHz ultrasonic waves to fill
Put, but it is not limited.
Above-mentioned generating device of laser, which can use, produces 300nm to the pulsed laser beam of 900nm wave bands, with 1W to 15W
Output, the pulse duration is 1ms to 900ms, and frequency is 1Hz to 100Hz laser aid, but not limited.
Temperature control equipment can be filled with temperature in use adjustable range in -40 DEG C to 99.9 DEG C of existing temperature control
Put, but it is not limited.
The pluripotent cell apparatus for deivation of the present invention, utilizes above-mentioned ultrasonic generator, generating device of laser or temperature control
Device processed, ultrasonic wave, laser or heat treatment are carried out to the mixture of culture medium and differentiated cell, pass through the training of a period of time
Support, form the orbicule with versatility, pluripotent cell can be divided into by differentiated cell is inverse.Now, in order to improve inverse point
Change efficiency, ultrasonic wave, laser or heat treatment first can be carried out to culture medium before mixed culture medium and differentiated cell.
Hereafter will according to embodiment, the present invention is described in detail, still, following embodiments be only the present invention example,
Present disclosure is not restricted to following embodiments.
Embodiment
<Embodiment 1>The preparation of Physics cells
Fig. 1 be the present invention Physics (pluripotent sphere yielded by ultrasonic sTimulus) cell forms simulation drawing, with 5W/cm2The ultrasonication ES of 10 minutes (embryonic stem) of intensity
Human dermal fibroblasts (HDFa, Cat.No.C-013-5C, GIBCO (Invitrogen cell are mixed in culture medium
culture))(1×106), use 1W/cm2The ultrasonication of intensity contains the mixture 5 seconds of cell.Screen the cell of existence
Afterwards, in the culture 2 × 10 that in petri diss (Petri dish), suspended in mankind's ES culture mediums of 35mm bacteriums5HDF 6
My god.
Cultivate the 1st day and initially form orbicule, non-differentiation marker thing is expressed after 3 days.
<Experimental example 1>The optimum condition experiment that orbicule is formed
Human dermal fibroblasts form orbicule in ultrasonication, in order to determine to be formed for improving orbicule
The optimum condition of efficiency, change ultrasonication condition, cell culture mode etc. and tested.
Cell culture mode on surface using not having in cated Tissue Culture Dish (bacterium petri diss) and train
Foster suspension culture (Suspended culture), and have coating on surface, cell is more easy to adhere to the Tissue Culture Dish on surface
The monolayer cultivation (Monolayer culture) of culture in (tissue culture dishes).
The control group (Null) of any processing is not carried out, ultrasonication is carried out to culture medium in addition, control group is divided into
Control group (UM:Ultrasound treated Media, use 5W/cm2The ultrasonication of intensity 10 minutes), to cell carry out
Control group (the UC of ultrasonication:Ultrasound treated Cell, use 1W/cm2The ultrasonication of intensity 5 seconds)
With the control group (UCUM that ultrasonication is carried out to both cell and culture medium:UM+UC), observe with the thin of incubation time change
Born of the same parents' form, the number of orbicule is determined, the orbicule number and size changed with incubation time is analyzed, so that it is determined that the spherical bodily form
Into efficiency.Experimental subjects cell is human dermal fibroblasts.
First, in order to determine ultrasonic intensity condition, HDF (1 × 10 is made6) be directly exposed under different ultrasonic intensities
(5 seconds, 0,0.5,1,3,5,10W/cm2).After the cell for screening existence, in 35mm bacterium petri disses, in mankind ES
2 × 10 are cultivated in culture medium5HDF 6 days.
【Table 1】ES medium components
As shown in Fig. 2 a (a), in 0.5,1 and 3W/cm2Ultrasonic intensity under, most of HDF of ultrasonication from
Aggegation is sent out, forms many cells orbicule.Control group is then attached to culture dish surface, with 5 and 10W/cm2The ultrasonication of intensity
HDF do not form orbicule, Apoptosis increases.
Fig. 2 a (b) shows the number of many cells orbicule generated in Fig. 2 a (a) under different ultrasonic intensities.
In 1W/cm2Ultrasonic intensity under live/dead cell analysis and image analysis result, as illustrated in figures 2 c and 2d, 25%
Cellular portions sustain damage, more than 95% cell keeps survival ability.However, than 1W/cm2Higher ultrasonic intensity
Under, HDF is caused Apoptosis by major injury.
Therefore, in 1W/cm2Mounting ultrasonic strength condition under, change open-assembly time (0,1,2,5,10,20,40 second),
In 35mm bacterium petri disses, cultivated 3 days in Human ES cells' culture medium.
As shown in Fig. 3 a and 3b (a)-(d), compared with other open-assembly times, when being exposed 5 seconds under ultrasonic wave, generation
Orbicule number highest.However, when exposing more than 10 seconds, Apoptosis dramatically increases, it is believed that this is because of cell membrane damage
It is caused.
Then, change ultrasonic wave exposure strength (0,1,5,10W/cm2), processing ES cell culture mediums 10 minutes.In 35mm
In bacterium petri diss, 2 × 10 under culture ultrasonic wave exposure5HDF(1W/cm2, 5 seconds) and 3 days.
As shown in figure 4, and 1W/cm2Compare, 5W/cm2In the culture medium of ultrasonication, about 2 times of orbicule is formed.
The change of open-assembly time (0,5,10,20 point) does not produce beneficial effect to orbicule formation efficiency.Typically
For, short open-assembly time can cause certain size range and more numbers (Fig. 5).
Then, after changing condition of culture, in order to verify that orbicule forms effect, ultrasonication is carried out to ESC culture mediums
(5W/cm2, 10 points), to HDF (1 × 106) carry out ultrasonication (1W/cm2, 5 seconds).Screen the HDF (1 × 10 of existence6) after,
In bacterium with carrying out suspension culture in Pi Shi culture mediums, or monolayer cultivation is carried out in tissue culture medium (TCM).
As shown in fig. 6, the HDF of the ultrasonication for the culture that suspends compared with monolayer cultivation, is shown higher spherical
Body formation efficiency.In addition, when ultrasonic stimulation applies to both cell and culture medium and stimulated, the higher spherical bodily form is shown
Into efficiency.
When suspending culture or monolayer cultivation to observe, the size of many cells orbicule of different ultrasonic stimulations generation
Distribution, in bacterium petri diss or tissue culture dishes, cultivated in ultrasonication or untreated ES culture mediums super
The HDF of sonicated or untreated HDF.
As shown in Figure 7 and Figure 8, in two kinds of culture dishes, HDF and culture medium are all observed by (UCUM) during ultrasonication
Higher orbicule formation efficiency.
Also, condition of suspension culture shows higher efficiency, the number and size of orbicule are bigger (more than 200 μm
Diameter), compared with monolayer culture conditions, show certain Size Distribution.
The HDF (UC) of the ultrasonication grown in untreated ES cell culture mediums forms orbicule.However, with
UCUM conditions are compared, and the number and size of orbicule are very low (to 200 μm).Trained in the ES cell culture mediums of ultrasonication
When supporting normal HDF (UM), a small amount of orbicule of small size (less than 100 μm) is formed.Utilize the monolayer cultivation of tissue culture dishes
UC and UM conditions, orbicule formation efficiency are very low.Most HDF is attached to culture dish surface, and orbicule number is considerably less.
In addition, when suspending culture or monolayer cultivation to analyze, many cells orbicule of different ultrasonic stimulations generation
Incubation time and representational undifferentiated gene expression relation, according to the method for above-described embodiment 1, by incubation time (1,
2nd, 3,4,5 and 6 days) control group and the cell of ultrasonication group (Null, UM, UC, UCUM) are reclaimed, use
MRNA direct kit (ambion) extract mRNA, after synthesizing SuperScrip-II (invtrogen) cDNA, using in table 2
The primer of record, is expanded by PCR, carries out electrophoretic analysis.
Fig. 9 is shown in by the RT-PCR results analyzed, HDF and culture medium are both by (UCUM) during ultrasonication, not
Differentiation marker gene is stable to express, and particularly, the culture that suspends is expressed higher compared with single-layer culturing cell.
In order to determine the orbicule of the undifferentiated attribute relevant with culture environment formed difference, compare suspension culture or
Non-differentiation marker thing OCT3/4 expression during monolayer cultivation.Therefore, will after ultrasonication culture a period of time (0,1,2,
3rd, 4,5 and 6 days) cell fix 30 minutes with 4% paraformaldehyde, in order to improve the penetrating power of antibody, added with 0.1%
In Triton X100 PBS after exposure 40 minutes, in order to prevent nonspecific proteins from reacting, using non-added with 5%
The PBS of lowlenthal serum carries out the closed process of 30 minutes at room temperature.Afterwards, after washing cell, add respectively once
Antibody (OCT4；1:200, abcam), in 4 DEG C of reactions overnight, washed using the PBS added with 0.03%Triton X100
After washing three times, secondary antibodies (IgG anti-rabbit conjugate alexa 488) are diluted respectively with D-PBS buffer solutions
To 1:1000, dye 2 hours at room temperature.Utilize the thin of the PBS washing dyeing added with 0.03%Triton X100
After born of the same parents four times, sealing solution (mounting sol.) of the sprinkling added with DAPI on slide, covered, using referring to
Behind nail polish sealing corner, observed with laser scanning co-focusing microscope.
As shown in Figure 10, it is interesting that ultrasonication may be immediately observed that OCT3/4 expression under the conditions of UCUM after 1 day.
Gradually increase, condition of suspension culture observe higher expression than monolayer culture conditions for OCT3/4 expression.
Afterwards, by culture culture orbicule 6 days under the conditions of UCUM that suspend, RT-PCR is utilized to analyze six kinds of undifferentiated marks
Remember that thing gene OCT3/4, SOX2, NANOG, TDGF1, c-MYC and KLF4 result are as shown in figure 11, OCT3/4 in 1 day after processing
With NANOG gene expressions increase, afterwards, the expression of other genes increases also with the increase of incubation time, is observed in 2 days
To the expression of all marker genes, stable expression is observed after 3 days.The time point of earliest OCT3/4 expression is in ultrasonic wave
Processing observes (Figure 12) after 10 hours.
In order to determine the undifferentiated ability of Physics cells, the RT-PCR results of the orbicule of 4 kinds of random screenings determine
Multipotency including OCT3/4, SOX2, NANOG, REX1, TDGF1, FOXD3, FGF4, UTF1, ESG1, LIN28a, KLF4, c-MYC
The expression of property marker gene, compares mankind H9ESC and normal HDF (Figure 13).Experiment includes recovery and cultivated 5 days
Physics cells, utilizeMRNA direct kit (ambion) extract mRNA, synthesize SuperScrip-II
(invtrogen) after cDNA, using the primer described in table 2, expanded by PCR, carry out electrophoretic analysis.
【Table 2】
Alkaline phosphatase (AP) coloration result shows the feature of the pluripotent state of many cells orbicule.Suspend culture
Orbicule is shown obvious red (Figure 14) compared with the orbicule of monolayer cultivation.OCT3/4、SOX2、NANOG、SSEA-4
Expression with TRA-1-60 is similar with H9 Human ES cells (Figure 15).Also, it is determined that many cells are spherical by flow cytometer
The versatility (Figure 16) of body.More than the 99.5% of SSEA-4 and TRA-1-60 is expressed in orbicule, each expression and H9 people
DX-like centers are similar.
In addition, transfer orbicule is sought in the tissue culture dishes for have gel coat with MEC (MEF)
When supporting cell co-culture, it was observed that cellular invasion and growth.Also, the expression of pluripotency marker's thing gene still keeps remaining unchanged
(Figure 17).
In addition, determine that the expression of non-differentiation marker thing has carried out DNA methylation analysis to add.Start in gene expression
Startup subdivision methylate, it is known that there is no gene expression in this section, the situation of demethylation, i.e. methyl from
The situation that DNA departs from, it is meant that the gene of this part has expression.Therefore, by determining that the oligogene of ES cells is i.e. undifferentiated
Whether the startup subdivision of both genes of the oligogene OCT3/4 and NANOG of stem cell methylates, and determines both genes
Whether express.
Therefore, utilize EZ DNA methylation kits after Physics orbicules extract DNA using Proteinase K and phenol
(Zymo Reaserch) analyzes the OCT3/4 and NANOG of Physics orbicules DNA methylation.Used in above-mentioned analysis
DNA cloning primer it is as follows：
1) mankind NANOG amplification primers：
Forward primer：5'-TAGGAGTAGAGTGTAGAGGAGAATGAGTTA-3'
Reverse primer：5'-ATCTATCCCTCCTCCCAAATAATC-3'
The size of amplified production：377bp, Tm:55, CpGs in product:6
2) mankind OCT4 amplification primers：
Forward primer：5'-TTTTTTTAAATTAGAAATTTTAATTATTTG-3'
Reverse primer：5/-AATTACAAAAACCATACCTACAACC-3'
The size of amplified production：417bp, Tm:55, CpGs in product:4
In the promoter region of the versatility evaluated by bisulfite sequencing-special OCT3/4 and NANOG genes
Cytosine-guanine dinucleotides (CpG) methylates, Physics cells height demethylation similar with ES cells, and original
HDF cells in, the CpG dinucleotides in this region then low demethylation (Figure 18).These results represent OCT3/4 and
NANOG promoters are activated by ultrasonication.
<Embodiment 2>The multiplication capacity of Physics cells and more differentiation capabilities
By proliferation marker albumen Ki-67 immunostainings and utilize hoechst (Hoechst 33342) and iodine
Change the multiplication capacity of the time difference nuclear targeting evaluation Physics cells of the third pyridine (PI).
As shown in figure 19, the expression of Ki-67 in Physics cells was detected at the 5th day.
In order to more clearly prove the multiplication capacity of Physics cells, prove that cell is bred by following methods.Using oozing
The Hoechst 33342 that permeability can dye well the nucleus of Survival Cells dyes to the Physics cells of culture the 5th day,
After removing staining reagent completely, cultivate 3 days, after the Physics cells of total culture 8 days are fixed with 4% paraformaldehyde, recycle
PI dyes to nucleus.Unduplicated danger signal means that to form new Physics thin because of cell division after 5 days
Born of the same parents.In addition, cultivating single orbicule 5 days, orbicule diameter is determined by image, proved by the increase of orbicule diameter dimension
The multiplication capacity of Physics cells.
In order to evaluate the self-regeneration ability of Physics cells, the Physics cells that will be dyed with Hoechst 33342
Additional culture 5 days, after being fixed with 4% paraformaldehyde, PI and OCT3/4 is recycled to dye again.PI signals represent that Physics is thin
Intracellular core.The cell kernel representation Hoechst 33342 almost redyed with the OCT3/4 merged of Hoechst 33342 is right before 5 days
Physics cells are dyed.
As shown in figure 20, during adding culture 5 days, versatility (OCT3/4) is not delivered to sub- Physics cells.These knots
Fruit represents that Physics cells can breed, but without self-regeneration after 5 days.
In addition, in the early stage cultivation period of Physics cells, the table of specific marker thing gene is found that in each germinal layer
Reach.The unique gene expression pattern for expressing the Physics cells of both undifferentiated and differentiation labels can be with the mankind
The EB in ESC sources is compared (Figure 21).Because their form is closely similar.
Immunocytochemical assay result finds Physics cells and the high expression endoderms (GATA4 and AFP) of EB, outer embryo
The label of leaf (PAX6 and Nestin), mesoderm (Brachyury and SMA).Except OCT3/4, PAX6 expression is also in ultrasound
Detect within first day after ripple processing.The expression of other genes of 3 germinal layers then starts for the 3rd day after Physics Hemapoiesis.
During culture 15 days, the expression of 3 germinal layer labels gradually increases.But OCT3/4 expression reduced (figure after 8 days
22)。
<Embodiment 3>Cellular change caused by ultrasonic stimulation
, to HDF effect, different ultrasonic wave bars is compared in order to evaluate ultrasonic stimulation during Physics cells are formed
Part.After ultrasonication and after the HDF2 hours of culture ultrasonication, sem analysis is directly carried out.
As shown in figure 23, several cell membrane stomatas are all formd under the conditions of UC and UCUM.It is probably because HDF directly exposes
The reason under ultrasonic wave.However, under the conditions of UM, HDF does not form any stomata by cell membrane.Because only to training
The reason for foster base carries out ultrasonication, and the damage to cell membrane is insufficient.Particularly, after cell culture 2 hours, the gas of generation
Hole all disappears under the conditions of UC and UCUM.These results mean that ultrasonic stimulation does not arrive the journey of inducing cell apoptosis seriously
Degree, but it is enough the of short duration infiltration of inducing cell film, so as to which during early stage cell culture afterwards, impaired cell membrane obtains
Recover.
The recovery process of the cell membrane of damage passes through also with live/dead cell detection kit (live/dead kit)
The analysis for the cell (red fluorescence) that cell membrane sustains damage without cell (green fluorescence)/death of damage or cell membrane is able to
Checking.The result that the fluorometric reagent for HDF cells carry out immediately and after 2 hours after ultrasonication dyes, 2 hours
The ratio of red fluorescence reduces afterwards, and sem analysis result is also the same, and the cell membrane damage caused by ultrasonic wave is able to after 2 hours
Recover (Figure 24).
In addition, in order to determine the relation of cell and the orbicule formation by ultrasonic stimulation, using reagent, by ultrasonic wave
The HDF of processing is added to live/dead cell detection kit, the HDF using living cells imaging device to green/red double staining
Follow the trail of within 24 hours.
As shown in figure 25, HDF is formed more with other only green dyeing or red/green double staining HDF aggegations
Spheroids.After 24 hours, the HDF of most slight damage forms stable Physics cells.
In addition, ultrasonic wave induction cell membrane damage and it is of short duration infiltration be also utilized respectively fluorescent dye Fluo-4 dyestuffs and
CM-H2DCFDA, according to increased intracellular Ca2+Concentration and intracellular H2O2Generation characterized.One is exposed to ultrasonic wave
Under, the Ca of Physics cells2+Concentration increases suddenly, then reduces (Figure 26) at 150 seconds.The intracellular H of Physics cells2O2
Compared with untreated control group HDF, ultrasonic wave improves 6 times (Figure 27 and Figure 28) concentration again after exposing 60 points.
Additionally, response various kinds of cell stress reaction in, by the use of ATP as signal, analyze to extracellular release
ATP concentration.
As shown in figure 29, compared with untreated HDF, the ATP in ultrasonic stimulation Physics cells discharges 22 times more.
The known expression for promoting ionic P2X acceptors and metabolic P2Y acceptors because of ATP release of active, therefore compare both by
The expression of body.
Detect that the expression of P2X4, P2X7, P2Y1, P2Y2 and P2Y11 in Physics cells are higher (Figure 30).
Additionally, the cell for the Physics cells for being determined to improve using the quantum dot (QD705) of Alexa-705 marks is inhaled
Receive.QD705 is added in culture dish, confocal images are obtained after 24 hours.Physics cells in orbicule type
The single cell type with adjacent single Physics cell agglutinations does not all absorb QD705.However, normal HDF does not inhale
Receive QD705 (Figure 31).This, which demonstrates ultrasonic stimulation, improves absorption of the cell to external key elements.
On the other hand, excretion body RNA is prepared by Physics cell culture mediums, it is thin by RT-PCR analysis and research Physics
The gene expression pattern of cell culture environment during born of the same parents are formed.Usually, excretion body includes several gene elements, for example, RNA,
MicroRNA, DNA, protein.In addition, the expression curve of gene element is cell state dependence in excretion body.
As shown in figure 32, pluripotency marker's thing gene is observed in the excretion body purified by Physics cell culture mediums
High expression.Most noticeable gene expression is OCT3/4 and NANOG.With the increase of incubation time, OCT3/4 expression
Dramatically increase.NANOG expression declines after 4 days.C-MYC expression keeps certain under condition of suspension culture, in monolayer cultivation bar
Reduced after 2 days under part.All pluripotency marker's thing genes, for example, REX1, TDGF1, FOXD3, UTF1 and LIN28, although
Expression is low, but is detected under condition of suspension culture.However, these genes do not detect under monolayer culture conditions
Arrive.These results mean the transmission possibility of gene element in the HDF of ultrasonication.
It is above-mentioned it is assumed that by the HDF without ultrasonication and Physics cell co-cultures in order to verify.In order to carry out
Graphical analysis, HDF is set to catch Cy5.5 red fluorescence dyestuffs using cationic-liposome (Lipofectamine).Physics is thin
Born of the same parents generate in addition, after being kept for 2 days, Physics cells are added in the HDF culture dishes of Cy5.5 colorings.During co-incubation,
Ultrasonication is not carried out to culture dish.Because UM conditions also can the expression of inducing pluripotent marker gene.
In confocal images, it was observed that with during Physics cell co-cultures, Cy5.5 coloring HDF
In have OCT3/4 expression.Do not detect that OCT3/4 is expressed in the HDF of Cy5.5 colorings is individually cultivated.These results are strong
Ground illustrates the versatility of Physics cells to adjacent normal cell transmission, and reprogramming is thin for the Physics of normal cell afterwards
Born of the same parents.In general, excretion body participates in the gene element transmission (Figure 33) in cell.
<Embodiment 4>The vitro differentiation of Physics cells
For vitro differentiation, the Physics cells cultivated 5 days are transferred in the tissue culture dishes of gel coat.Profit
Cell differentiation with the induction transfer of specific differentiation culture medium is nerve or cardiac system.Above-mentioned specific differentiation culture medium such as table
Shown in 3.Found in the cell of 8 days after induction is broken up 3 germinal layer main proteins (GATA4, AFP, PAX6, Nestin,
Brachyury, SMA) expression (Figure 34).
【Table 3】
【Table 4】
As shown in Figure 35 and Figure 36, within the differentiation period of 1-2 weeks, SRY-box expression, SRY- are observed by RT-PCR
Box includes gene 17 (SOX17, endoderm), paired box 6 (PAX6, epiblast), Nestin (nerve cell label), micro-pipe
GAP-associated protein GAP 2 (MAP2, epiblast), type III beta tubulin (TuJ1, nerve cell label), MSH homologous proteins 1 (MSX1,
Mesoderm), Brachyury (mesoderm), myosin light chain 7 (MYL7, cardiac muscle cell), NK2 homologous proteins 5 (NKX2.5, the heart
Myocyte) and TnT 2 (TnnT2, cardiac muscle cell).
Particularly, OCT3/4 expression is valuably reduced after induction.
Or the differentiation to nerve or heart cell is determined by immunocytochemical method.
As shown in Figure 37 to Figure 39, neural precursor is observed in the Physics cells grown in growing cell culture medium
Cell marker (PAX6 and Nestin).Growth cell culture medium is converted into oligodendroglia culture medium or nerve cell training
After supporting base, these Physics cells broken up are induced to break up time of 2 weeks again.Respectively it was observed that oligodendroglia marks
The expression of thing (MAP2 and O4) or nerve cell label (MAP2 and Tuj1).The divergaence time of 2 weeks to detection include MHC,
Cardiac marker including SMA, Actinin, NKX2.5 and TnTc is sufficient.Particularly, detected in actinine
The actin pattern of typical merogenesis.However, under identical condition of culture, HDF does not express any nerve or heart mark
Remember thing.
<Embodiment 5>Stability test
Ultrasonic wave does not have the undesirable side effects such as induced mutation, gene alteration, cancer generation.Physics cells have just
Normal caryogram (Figure 40).
<Embodiment 6>Differentiation potency is evaluated inside Physics cells
In order to evaluate differentiation capability inside Physics cells, the Physics cells 1 × 10 of 5 days will be cultivated6Individual injection
In the testis and leg muscle of the immunodeficient mouse (NOD/SCID mouse) of 4-5 weeks, after raising 4 weeks, testis and flesh are reclaimed
Meat, after being fixed with 4% paraformaldehyde, freezing microtome section (cryosection), the cell for judging to inject is dyed by people's nuclear antigen
Position, dyeing is a variety of with propagation and the related protein labels (Ki67, CD44, SMA) of differentiation, it is determined that the cell injected whether
Differentiation.
In Figure 41, the Physics cells for injecting testis are observed in intratesticular vascular endothelial cell after 4 weeks,
Have propagation so as to dye determination by Ki67, as illustrated, with the cell of arrows it was observed that vascular endothelial cell
Label CD44 is colored.
The cell injected to mouse thigh is found, so that it is determined that muscle egg outside muscle fiber layers in lamella
White matter label SMA is colored (Figure 42).Mean that the cell adapted peripheries of Physics that are injected in vivo are thin with this identical result
Born of the same parents and environment are broken up.
<Embodiment 7>The effect of cell culture medium
Physics cells are formed using Human ES cells' culture medium.ES cell culture mediums are opened as defined medium
Hair, for ES cells to be kept and bred under undifferentiated state.In order to study the effect of cell culture medium, trained using normal HDF
Support base and form Physics cells.
As shown in Figure 43 a and 43b, compared with ES cell culture mediums, form and orbicule formation efficiency have different.
After the HDF inoculation of mediums of ultrasonication, first day a small amount of formation many cells orbicule.However, after 2 days, it is most
Orbicule is attached to culture dish surface.When cultivating the 4th day, all orbicules are attached to media surface, grow into typically into
Fibrocyte form.Immunocytochemistry result shows different gene tables between also showing two kinds of different culture medium conditions
Expression patterns.Using ES cell culture mediums generation typical Physics cells in OCT3/4, SOX2, NANOG, SSEA-4 and
TRA-1-60 expression is high.DMEM culture mediums do not show any for inducing non-differentiation marker thing gene and 3 germinal layers
The effect of marker gene expression.These results mean that orbicule is formed and the expression of specific marker thing gene is trained with cell
The composition for supporting base is closely related.
<Embodiment 8>The effect of cell line
In order to evaluate whether Physics cells forming method is applied to other cell lines and whether be applied to later clinic
Using utilizing other cell lines such as HeLa cells, L132 mankind's pulmonary epithelial cells and skin fibroblasts from patient
Study the formation of Physics cells.
As shown in Figure 44 a-44c, make after 2 kinds of cell lines are directly exposed under ultrasonic wave, in bacterium petri diss
In cultivated in the ES cell culture mediums of ultrasonication, formed many cells orbicule.It is interesting that generated with HDF
Physics cells compare, and the form and Size Distribution of the new Physics cells of this 2 kinds of cell line generations have different.HeLa
The Size Distribution of the Physics cells of cell derived does not have consistency quite, and size is too big.The Physics of L132 cell deriveds
Cells show goes out the form of more complicated aggegation.Each orbicule adds fusion and forms platy structure.
As shown in Figure 44 b and 44c, determine what is expressed by 2 kinds of other Physics cells by immunocytochemical method
The pluripotency marker such as OCT3/4, SOX2, NANOG, SSEA4 and TRA-1-60 thing and GATA4, AFP, PAX6, Nestin,
The germinal layer marker genes of Brachyury and SMA etc. 3.
In addition, also forming the orbicule similar to Physics cells in the experimental result using patient skin cell, lead to
Cross the expression (Figure 45) that immunocytochemical method determines pluripotency marker's thing and 3 germinal layer labels.
These results forcefully demonstrate the induction plastidogenetic ultrasonic stimulations of Physics go for it is a variety of thin
Born of the same parents are, so as to show the possibility for utilizing the cell Heal Thyself of Patient cells.
<Embodiment 9>The effect of other energy sources
Applying additional external to HDF and ES cell culture mediums stimulates, it is determined that and evaluating the Forming Mechanism of Physics cells.
Ultrasonication is replaced with laser treatment and determines Physics cells either with or without formation.Therefore, same use is in ultrasonication
When used human dermal fibroblasts, laser treatment condition is to use Ocla treatments laser (Ndlux), irradiation
808nm laser is cultivated after 5 seconds.
In order to be heat-treated, skin fibroblasts is exposed at 42 DEG C after 2 minutes, about 5 seconds are stood on ice.
As shown in Figure 46 and 47, after laser or heat treatment are carried out to both HDF and ES cell culture mediums, have successfully formed
Many cells orbicule.The HDF of laser treatment is also to form many cells orbicule immediately after induced with laser.Although the shape of orbicule
Shape is irregular, and Size Distribution is uneven, but also observes pluripotency marker's thing and 3 germinal layer label high level expressions.At heat
The same induction orbicule of reason is formed.However, efficiency is lower than ultrasonic wave and laser treatment.The more of thermal induction are utilized during being kept for 8 days
More than half of spheroids is attached to culture dish surface.Although orbicule formation efficiency is lower, it has been observed that versatility
The expression of label and 3 germinal layer labels is high.These results forcefully demonstrate formation and the physics of Physics cells
Stimulate closely related.
<Embodiment 10>Prepare mouse Physics cells
Process according to Figure 48 prepares mouse Physics cells.Therefore, with 20KHz ultrasonic waves with 5W/cm2's
Intensity handles in the ES culture mediums of 10 minutes and mixes OG2 mouse MEF (Mouse Embryonic Fibroblast cell：Mouse
Embryo fibroblast), to above-mentioned cell direct irradiation ultrasonic wave, with 1W/cm2Intensity handle and cultivated for 5 seconds.Utilize
Fluorescence microscope, the cellular morphology change and the expression of GFP fluorescence of the cell of culture are observed with the interval of 1,3,5,8 and 10 day.
Medium component for ultrasonication is as shown in table 1.
Above-mentioned MEF cells are the big embryos on the 13.5th of the mouse for the GFP genetic transformation for making insertion OCT4 promoters into fibre
Cell is tieed up, without the cell of expression, GFP is expressed usually OCT4 if OCT4 is expressed, and shows green fluorescence.
In Figure 49 A, without presentation green fluorescence in the OG2MEF cell images of control group (OCT4 is not expressed).However,
The OG2MEF of ultrasonication situation is carried out, with the size increase of the increase spheroids of incubation time, it is known that
The intensity enhancing of green fluorescence.This represents that ultrasonication is expressed induction of OCT4, and OCT4 is the main spy of undifferentiated stem cell
Sign, it can therefore be appreciated that ultrasonication causes, OG2MEF cells are inverse to be divided into stem cell.
Figure 49 B are tile scan images, the image synthesized after multiple images for being shooting wide scope, are shown at ultrasonic wave
Effect is managed, most MEF cells show the ball of generation caused by ultrasonication in inverse expression of differentiation OCT4-GFP, Figure 50
The GFP expression efficiency analysis results of shape body, about 93% or so OCT4-GFP expression efficiencies are shown, it is true using flow cytometer
Determine the result that GFP is expressed in whole cell, GFP has expression in about 85.3% cell, analyzes the undifferentiated albumen of cell surface
Matter label SSEA1 expression result be also have about 75.5% expression (Figure 51).These results mean caused by ultrasonic wave
Inverse differentiation efficiency is very high.
Afterwards, using the primer sets shown in table 5 below, determined by RT-PCR and immunocytochemistry method representational
The non-differentiation marker thing gene of mouse embryo stem cell (ESc) and protein label (OCT4, SOX2, NANOG, SSEA1)
Expression.
As shown in figs. 52 and 53, it is determined that have the expression of non-differentiation marker thing in mouse Physics cells.
In addition, also it is determined (Figure 54) by alkaline phosphatase staining
【Table 5】For determining the RT-PCR primers of mouse ES non-differentiation markers thing expression
【Table 6】For determining the RT-PCR primers of mouse differentiation marker expression
Confirm the result of 3 germinal layer labels, mPhysics makes endoderm (GATA6), epiblast (Nestin), mesoderm
(Brachyury) the high expression of label.The expression of other genes of 3 germinal layers starts for the 3rd day after Physics cells are formed.
During culture 20 days, the expression of 3 germinal layer labels gradually increases (Figure 55).It is also, true by immunostaining in Figure 56
The expression of 3 germinal layer protein labels is determined.
In mouse cell, because ultrasonic waveform into mPhysics cells also there is normal caryogram (Figure 57).
Industry application possibility
The pluripotent cell of the present invention can be used for cellular therapeutic agent field.
Claims (18)
1. it is a kind of by the inverse method for being divided into pluripotent cell of differentiated cell, including：
The differentiated cell is mixed with culture medium；With
By forming orbicule to mixture offer energy and by the mixture culture scheduled time,
Wherein, the orbicule has versatility feature.
2. according to the method for claim 1, it is characterised in that the energy includes being selected from ultrasonic wave, laser and heat treatment
In any one.
3. according to the method for claim 1, it is characterised in that orbicule expression selected from OCT3/4, SOX2, NANOG,
It is any in c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4 and AFP
A kind of non-differentiation marker thing gene or 3 germinal layer marker genes.
4. according to the method for claim 1, it is characterised in that the differentiated cell is included selected from mammal source
Any one in fibroblast, cancer cell or organ inner tissue cell.
5. according to the method for claim 1, it is characterised in that the culture medium include selected from embryonic stem cell medium and
Any one in stem cell induction culture medium.
6. method according to claim 1 or 2, it is characterised in that be additionally included in culture medium and differentiated mixing with cells
Before, it is 1W/cm with output intensity2To 20W/cm2Ultrasonication described in culture medium 1 to 20 minute.
7. method according to claim 1 or 2, it is characterised in that to the culture medium and the mixture of differentiated cell
It with output intensity is 0.5W/cm that the ultrasonication of progress, which is,2To 3W/cm2Ultrasonication 1 to 5 second.
8. method according to claim 1 or 2, it is characterised in that be additionally included in culture medium and differentiated mixing with cells
Before, the culture medium is irradiated 1 to 20 second with 300nm to 900nm wave bands pulsed laser beam.
9. method according to claim 1 or 2, it is characterised in that to the culture medium and the mixture of differentiated cell
The laser treatment of progress is irradiated 1 to 10 second with 300nm to 900nm wave bands pulsed laser beam.
10. method according to claim 1 or 2, it is characterised in that be additionally included in culture medium and differentiated mixing with cells
Before, the culture medium is heat-treated under 40 DEG C to 50 DEG C of temperature conditionss 5 to 20 minutes.
11. method according to claim 1 or 2, it is characterised in that to the culture medium and the mixture of differentiated cell
The heat treatment of progress is after the mixture is exposed 1 to 10 minute under 40 DEG C to 50 DEG C of temperature conditionss, at 0 DEG C to 4 DEG C
Temperature conditionss under exposure 5 to 10 seconds.
12. according to the method for claim 1, it is characterised in that will be provided energy with suspension culture or monolayer cultivation mode
The mixture culture of amount 3~10 days.
13. a kind of pluripotent cell apparatus for deivation, including：
For storing the culturing room of differentiated cell and culture medium；With
It is arranged on the side of the culturing room, the power supply device of energy is provided to the differentiated cell and the culture medium,
Wherein, the differentiated cell and the culture medium are mixed, by providing energy to obtained mixture and mixing this
The compound culture scheduled time forms orbicule, and
The orbicule has versatility feature.
14. pluripotent cell apparatus for deivation according to claim 13, it is characterised in that the culturing room is by realizing the training that suspends
Support or the structure of monolayer cultivation mode is formed.
15. pluripotent cell apparatus for deivation according to claim 13, it is characterised in that the power supply device includes being used to send out
Penetrate the ultrasonic generator of ultrasonic wave, generating device of laser or temperature control equipment for launching laser.
16. pluripotent cell apparatus for deivation according to claim 15, it is characterised in that the ultrasonic generator transmitting
Frequency is 10kHz to 100MHz ultrasonic wave.
17. pluripotent cell apparatus for deivation according to claim 15, it is characterised in that the generating device of laser is sent
300nm to 900nm wave bands pulsed laser beam.
18. pluripotent cell apparatus for deivation according to claim 15, it is characterised in that the temperature control equipment is by temperature
Control is in the range of -40 DEG C to 99.9 DEG C.
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| CN109089423A (en) * | 2016-03-11 | 2018-12-25 | 斯特昂株式会社 | The cell reprogramming method flowed into using the environment as caused by physical stimulation |
| CN114264639A (en) * | 2021-12-23 | 2022-04-01 | 深圳大学 | Visualization device for inducing micro-damage of cells and fluorescence monitoring method |
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| KR101798726B1 (en) * | 2014-12-04 | 2017-11-16 | 가톨릭관동대학교기술지주 주식회사 | Device and method for inducing pluripotency cells yielded by energy |
| US20180127738A1 (en) * | 2016-11-07 | 2018-05-10 | BiomediStem, LLC | Production and therapeutic uses of epinul pluripotent cells and differentiated cells derived therefrom |
| KR101978270B1 (en) | 2017-12-27 | 2019-05-14 | 연세대학교 산학협력단 | Apparatus and method for mass transfer in the 3D cell culture |
| KR102416171B1 (en) * | 2018-10-02 | 2022-07-06 | 주식회사 스템온 | Reprosomes, the exosomes capable of inducing reprogramming of cells and the preparation method thereof |
| KR20240004541A (en) * | 2018-11-08 | 2024-01-11 | 이슘 리서치 디벨롭먼트 컴퍼니 오브 더 히브루 유니버시티 오브 예루살렘 엘티디. | Anchorage-independent cells and use thereof |
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| CN114264639B (en) * | 2021-12-23 | 2023-07-04 | 深圳大学 | Visualization device for cell micro-damage induction and fluorescence monitoring method |
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